Identification and partial characterization of potential probiotic lactic acid bacteria in freshwater Labeo rohita and Cirrhinus mrigala

Carps are the most diversified freshwater fish belonging to family Cyprinidae. Numerous probiotic and pathogenic lactic acid bacteria (LAB) have been characterized from carps. However, the diversity of these ecologically important bacteria is entirely unknown in freshwater fish of Pakistan. The present study aimed to characterize and identify the lactic acid bacteria from two carps viz. Laboe rohita and Cirrhinus mrigala and determine their antagonistic activity. Seventeen bacterial isolates were purified from the gastrointestinal tract and gills of these fish and characterized morphologically. Initially, seven isolates were screened as LAB using agar supplemented with CaCO₃. Subsequently, only two isolates CILB2 and RIL10 were selected as LAB after high‐performance liquid chromatography analysis for lactic acid production. Isolates CILB2 and RIL10 were genetically identified as Enterococcus faecalis and Weissella sp., respectively after 16S rRNA gene sequence analysis. Both strains exhibited significant antagonistic activity against common fish pathogens Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli. Enterococcus faecalis CILB2 and Weissella sp. RIL10 were also found negative for haemolysis and gelatinase activities and were sensitive to ampicillin, amoxicillin, doxycycline, erythromycin, chloramphenicol and co‐trimoxazole antibiotics. The identified LAB strains may further be investigated for their potential probiotic application in fish feed and food preservation techniques.     

A simple approach for regulating saline groundwater inflows to natural streams in irrigated agricultural areas

Stream salinity management is of prime importance for ensuring environmental sustainability of rivers, streams, lakes and other water bodies. Saline groundwater inflows are the main cause of deteriorating quality of stream flows; especially during periods of low flows. The Murray-Darling Basin is a good example for reducing groundwater recharge from irrigated agricultural areas with successful implementation of land and water management plans (LWMPs). In most cases, these plans help minimize the influence of saline groundwater flows on the stream salinity, as a preventive measure. As a remedial measure, this paper introduces a simple but an innovative approach for regulating saline groundwater inflows to natural streams in irrigated agricultural areas. The approach uses the flap gate with an automatic lowering and raising mechanism. During low stream flow, this gate is kept in a raised position. Depending upon the upstream inflows, the ponding situation will develop hydraulic gradient away from the stream; groundwater inflows to the creek will become minimal. During high stream flows, the gate is kept in a lower position to let the diluted water flows through the stream uninterrupted. To install, operate and manage such gates on multiple sites along the stream(s), the individual control panels can be joined into a central control station via telemetric link up. This approach, which holds a key for successfully regulating groundwater induced salinity to natural streams, should be considered in conjunction with other LWMPs to improve stream salinity in an irrigated agricultural area.     

Virulence-associated genes and antimicrobial resistance among avian pathogenic Escherichia coli from colibacillosis affected broilers in Pakistan

Avian pathogenic Escherichia coli (APEC) causes colibacillosis that leads to high morbidity and mortality among poultry birds. To date, there is a lack of knowledge about virulence-associated genes (VAGs) and multidrug resistance of APEC isolates from Pakistan. In this study, we determined the VAGs and antibiotic resistance profiles of APEC isolates recovered from colibacillosis affected broilers in Faisalabad region of Pakistan. A total of 84 diseased and dead birds from different local broilers farms were collected and examined for the gross lesions of colibacillosis by conducting postmortem examination. Of these, APEC isolates were recovered from 75 (89.2%) birds. Antibiotic susceptibility tests against 11 antimicrobial agents showed the highest resistance against ampicillin (98.6%) followed by tetracycline (97.3%) and ciprofloxacin (72%). The presence of 11 virulence-associated genes (VAGs) was detected by multiplex polymerase chain reaction (PCR). Of the 75 APEC, 32 (42.6%) harbored > 5 VAGs. Most commonly found genes were increased serum survival (iss; 84%), iron transport (iutA; 74.6%), and colicin V (ColV; 60%). Twenty-two isolates (29.3%) were found to possess a combination of VAGs; iss, tsh, iroN, and iutA, in addition to other VAGs. To the best of our knowledge, this is the first report on the detection of virulence-associated genes and multidrug resistance among APEC isolates in Pakistan. In the future, the strains with the predominant set of VAGs can be used for colibacillosis diagnosis and as a potential vaccine candidate.

     

Development of a two-antibody model for the evaluation of copper-64 radioimmunotherapy

Copper‐64 emits β⁺ and β^– particles suitable for positron emission tomography and radioimmunotherapy (RIT) of cancer. Copper‐64‐labelled antibodies have caused complete responses in laboratory animal RIT studies at far lower radiation doses than traditionally prescribed. The intracellular localization of copper radioisotopes may lead to cytotoxic effects by mechanisms beyond ionizing radiation damage. The purpose of this research was to develop a model using both internalizing and non‐internalizing antibodies for direct comparison in future RIT studies using the same animal model of cancer. The monoclonal antibodies, cBR96 and cT84.66, were conjugated with N‐hydroxysulfosuccinimidyl DOTA. All conjugates retained high immunoreactivity and labelled efficiently with ⁶⁴Cu with high specific activity and radiochemical purity. Twenty‐four hour biodistributions determined in LS174T tumour‐bearing nude mice demonstrated low organ and high tumour uptakes for both monoclonal antibodies. This model constitutes a promising system for elucidating whether internalization of ⁶⁴Cu is responsible for an enhanced tumour cytotoxicity in vivo.     

A two‐antibody model for copper‐64 radioimmunotherapy: biodistributions and tumor dosimetry in a mouse model of cancer

Objectives: We previously presented preliminary data on pilot biodistributions of ⁶⁴Cu‐labeled monoclonal antibodies (mAbs). 1 The purpose of the present studies was to obtain comprehensive biodistributions using a two‐antibody model for direct comparison of an internalizing and a non‐internalizing mAb in the same animal model of cancer. These studies will allow evaluation of the efficacies of the two ⁶⁴Cu‐labeled mAbs for radioimmunotherapy (RIT) and are part of a larger, ongoing project examining the unusual tumoricidal properties of internalized ⁶⁴Cu. Methods: DOTA (1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid) was conjugated to an internalizing antibody, cBR96, and a non‐internalizing antibody, cT84.66. Biodistributions of the ⁶⁴Cu‐labeled conjugates in nude mice bearing LS174T human colorectal carcinoma xenografts were obtained at time points from 15 min to 48 h. Mouse tumor dosimetry was calculated using a Monte Carlo N‐Particle Transport Code. Results: The ⁶⁴Cu‐DOTA‐cBR96 conjugate demonstrated rapid tumor uptake, reaching 20.2% injected dose per gram of tissue (% ID/g) at 3 h and peaking at 35.4% ID/g by 24 h. Tumor accumulation of ⁶⁴Cu‐DOTA‐cT84.66 was more gradual, 8.19% ID/g at 3 h (p = 0.002), and reached 43.8% ID/g by 24 h, but maximum uptake was not statistically different from ⁶⁴Cu‐DOTA‐cBR96 (p = 0.05). Liver, spleen, and kidney uptakes were statistically similar at 24 h for both conjugates (p = 0.05). Tumor absorbed radiation doses were estimated to be 1128 rad/mCi for ⁶⁴Cu‐DOTA‐cBR96 and 1409 rad/mCi for ⁶⁴Cu‐DOTA‐cT84.66. Conclusion: The biodistributions and tumor dosimetry of these two radiolabeled antibodies were sufficiently similar for direct comparison of the therapeutic efficacies of internalizing versus non‐internalizing ⁶⁴Cu RIT agents in the same animal model of cancer. RIT studies using this two‐antibody model are currently in progress to test the hypothesis that internalization of ⁶⁴Cu is necessary for enhanced cytotoxic properties at low tumor absorbed doses.     

Distribution of Trace Metals in Channel Sediment: a Case Study in South Atlantic Coast of Spain

Recently, Sancti Petri channel on the southwestern (SW) part of Iberian Peninsula has been experiencing urban, industrial, and vehicular expansion. Until recently, there have been only few published reports documenting the pattern of metal accumulation in this estuarine sediment. In the present study, trace metals such as Cu, Zn, Ni, Mn, Pb, Co, Cd, As, and Hg concentrations were analyzed from 69 sediment samples collected from 23 sampling sites of the Santi Petri channel. The magnitude of trace metal accumulation found as the following trend: Mn > Zn > Cu > Pb > Ni > Co > As > Cd > Hg. Spatial distribution pattern demonstrated overall decreasing trend of trace metal from Cadiz Bay mouth to the open ocean mouth, clearly correlative to the presence of anthropogenic inputs. Results of the principle component analysis (PCA) revealed that sediment metal chemistry of Sancti Petri channel is mainly regulated by the concentrations of Pb, Cu, Zn, and Ni; possible sources of those were from vehicular-related emissions. Pollution load index (PLI) and geo-accumulation index (I _geo ) indicated overall low values. The study will stimulate improvement of our understanding regarding the pattern of accumulation of metals in the coastal sediments, and the recorded values of metals in the present study can be used as suitable reference for future studies.     

Development of a SYBR Green-based real-time quantitative polymerase chain reaction assay to detect enzootic nasal tumor virus in goats

Enzootic nasal adenocarcinoma is a contagious respiratory disease in goats that is caused by the enzootic nasal tumor virus 2 (ENTV-2). In order to increase the number of available detection methods for ENTV-2, we developed a SYBR Green real-time polymerase chain reaction (SGrPCR) assay that targets the gag gene of ENTV-2. The low limit of detection of the assay was 3.68 × 10¹ copies/μL, a hundredfold more sensitive than conventional PCR. The melt curve showed a single sharp melt peak at 83°C, which indicated that there was no non-specific amplification or primer dimer formation. The intra-assay and inter-assay coefficients of variation were 1.58% and 1.82%, respectively. There was no cross-reactivity with closely related goat viruses (i.e., orf virus, peste des petits ruminants virus, goatpox virus, foot-and-mouth disease virus) and endogenous retroviruses. In conclusion, the SGrPCR assay is specific for the gag gene of ENTV-2 and provides a rapid and sensitive approach for detecting ENTV-2 in clinical samples.